Intracellular Proatherogenic Events and Cell Adhesion Modulated by Extracellular Thiol/Disulfide Redox State
http://www.100md.com
《循环学杂志》
the Department of Medicine, Divisions of Cardiology and Pulmonary, Emory University, Atlanta, Ga.
Abstract
Background— Oxidative stress, a contributing factor to atherosclerosis, causes oxidation of biological thiols, which can be quantified in terms of the thiol/disulfide redox. The major thiol/disulfide redox couple in human plasma is cysteine (Cys) and its disulfide, cystine (CySS). Although atherosclerosis has previously been associated with Cys/CySS oxidation, whether oxidation of Cys/CySS contributes in a causal way to atherosclerosis development is not known. We examined the function of extracellular Cys/CySS redox potential (Eh) in the regulation of early events of atherosclerosis using cultured aortic endothelial cells and monocytes as a vascular model system.
Methods and Results— To determine the range of thiol/disulfide redox state in human plasma, we analyzed levels of Cys, CySS, glutathione (GSH), and glutathione disulfide (GSSG) and calculated Eh according to the Nernst equation. Eh of Cys/CySS and GSH/GSSG was –120 to –20 and –200 to –50 mV, respectively. To approximate this range, endothelial cells were exposed to initial Eh from –150 mV (most reduced) to 0 mV (most oxidized). Compared with more reduced Eh, oxidized Eh of Cys/CySS stimulated H2O2 but not nitric oxide production, activated nuclear factor-B, increased expression of adhesion molecules (intercellular adhesion molecule-1, platelet endothelial cell adhesion molecule-1, P-selectin), and stimulated monocytes binding to endothelial cells. Extracellular Eh regulated thiol/disulfide redox states of extracellular membrane proteins and H2O2 production, indicating that variation in extracellular Eh is detected and signaled at the cell surface.
Conclusions— The extracellular thiol/disulfide Eh of the Cys/CySS couple plays a key role in regulating early events of atherosclerosis and could be useful as a potential marker for vascular disease risk.
Key Words: atherosclerosis ; cell adhesion molecules ; endothelium ; inflammation ; plasma
Introduction
Considerable evidence implicates oxidative stress and endothelial dysfunction and injury as initiation events leading to atherosclerotic cardiovascular disease.1–4 Atherosclerosis is a chronic immune-mediated disease; its initiation, progression, and destabilization are driven and regulated by inflammatory cells. Critical events in the initiation of the vascular inflammatory disease are the adhesion of leukocytes to the dysfunctional and injured endothelium and the penetration of these cells into the vessel wall to generate foam cells. Mechanistic studies show that transcriptional activation of adhesion molecules, including P-selectin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule (PECAM),5–7 by nuclear factor-B (NF-B)8,9 plays a pivotal role in the process of cell-cell adhesion. NF-B is activated by oxidative stress, which can be caused by reactive oxygen species (ROS) and reactive nitrogen species generated in free radical reactions from products of enzymes (NADPH oxidases and nitric oxide [NO] synthases [NOSs]) present in the vascular endothelium and smooth muscle.
Accumulating evidence indicates that redox-regulated biological signaling pathways respond not only to ROS but also to the redox state (Eh) of the thiol/disulfide couple.10–12 The reversible redox reactions of thiol/disulfide pools regulate diverse biological processes, including enzyme catalysis, gene expression, cell proliferation, and atherosclerosis.13–16 Most studies thus far have focused on the major cellular thiol, glutathione (GSH), and its disulfide (GSSG). Relatively less information is available concerning cysteine (Cys) and its disulfide, cystine (CySS), which together constitute the predominant low-molecular-weight thiol/disulfide pool in human plasma. Cys residues in proteins are critical for the function of many enzymes, receptors, ion channels, transporters, and transcription factors and are common targets of oxidation. Free cysteine undergoes rapid autoxidation and is toxic to mammalian systems at concentrations exceeding the normal, low-micromolar concentrations found in plasma because its oxidation results in generation of ROS. However, little is known about the effects of variation in Cys/CySS redox state, such as the progressive oxidation of Cys/CySS associated with increasing age17,18 and smoking,19 on cellular events related to atherosclerosis.
In the present study, we hypothesized that oxidation of the thiol/disulfide redox state over the range found in vivo in human plasma is a key determinant of early events of vascular disease development. To test this hypothesis, we compiled data from 4 previous studies of thiol/disulfide redox state in human plasma to determine the range of redox potential. Initial redox states spanning this range were then used with an in vitro model system to study early signaling events of monocyte adhesion to vascular endothelial cells. Results show that oxidized extracellular redox state of Cys/CySS (Eh, 0 mV) stimulated attachment of monocytes through mechanisms involving stimulation of H2O2 generation, activation of NF-B, and increased expression of adhesion molecules, including ICAM-1, PECAM-1, and P-selectin. These findings suggest that oxidation of extracellular Cys/CySS redox potential could play a key role in proatherosclerosis processes.
Methods
Cell Culture, Transfection, and Cell Treatment
Bovine aortic endothelial cells (BAECs) harvested from descending thoracic aortas and THP1 purchased from ATCC were maintained with 10% FBS (37°C, 5% CO2) in DMEM and RPMI, respectively. Before experiments with controlled initial redox states, medium was changed to cyst(e)ine-free DMEM or cyst(e)ine-free RPMI with 0.5% FBS. For transfections, BAECs (4 to 5x105 cells per plate) were grown overnight and transfected with FuGene 6 (Roche). Cells were exposed to different Eh for 1 day before the luciferase assay was performed. For transfections, cells were cotransfected with pNFB-Luc (BD science) or pTAL-Luc (BD science) and with pcDNA3.1 containing lacZ gene (Invitrogen). We obtained 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, disodium salt (AMS), monobromotrimethylammoniobimane bromide (qBBr), biotin-maleimide [N-(3-maleimidylpropionyl)biocytin], 6-carboxy-2', 7'-dichlorofluorescin diacetate (DCF-DA), and luminol from Molecular Probes; diphenyleneiodonium chlroride (DPI) was from Biolmol. Cys, CySS, catalase–polyethylene glycol (PEG), NG-nitro-L-arginine methyl ester hydrochloride (L-NAME), and NG-nitro-L-arginine (L-NNA) were from Sigma-Aldrich.
Assays
The luciferase activity was normalized for transfection efficiency as described previously.20 To generate the desired Eh, varied concentrations of Cys and CySS were added to cyst(e)ine-free media to give a constant total amount of Cys equivalents (200 μmol/L) as described previously.11 Cells were replenished with fresh medium every 10 hours. The medium and cell lysates collected for the analyses of Cys, CySS, GSH, and GSSG were derivatized, followed by high-performance liquid chromatography analysis as reported previously.15 Eh for Cys/CySS was calculated with the Nernst equation: Eh=E0+RT/2F ln([CySS]/[Cys]2), –250 mV for E0 at pH 7.4.15 To examine the effect of Cys/CySS redox on adherence of monocytes to BAECs, THP1 cells labeled by calcein AM (Molecular Probes)21 were added to BAECs at a cell density 1x106 or 5x104 per well for 6- or 96-well plates, respectively. The 96-well plates were incubated for 30 minutes (37°C, 5% CO2), washed 3 times to remove unbound THP1, and scanned by the plate reader to measure total fluorescence in each well. For ROS detection, dichlorofluorescin (DCFH) and luminol were used. To measure ROS production by DCFH oxidation, BAECs plated into 96-well plate were incubated with the Cys/CySS-limiting medium. BAECs were washed with KRH buffer,22 incubated with DCFH-DA at 50 μmol/L for 30 minutes (37°C, 5% CO2), and washed, and the fluorescence of cells from each well was measured by the plate reader. Additionally, ROS production in BAECs after Cys/CySS redox treatment was determined by monitoring chemiluminescence enhanced by luminol.
Determination of Extracellular Thiols and Western Blot Analysis
To determine extracellular thiols, BAECs plated in 96-well plate were exposed to Cys/CySS redox for 2 hours, washed twice with Hank’s balanced salt solution (HBSS), incubated with AMS (0.5 mmol/L) for 1 hour, and washed, and extracellular thiols conjugated with AMS were quantified by fluorescence (excitation, 324 nm; emission, 410 nm). To examine extracellular thiols present in the plasma membrane by Western blot analysis, BAECs grown in 150-mm plates were exposed to Cys/CySS redox for 2 hours, washed with HBSS, incubated with thiol-reactive biotinylation reagent (biotin maleimide, 0.5 mmol/L) for 1 hour, washed, lysed with a lysis buffer (1 mmol/L NaHCO3, pH 7.2, 2 mmol/L CaCl2, 5 mmol/L MgCl2, 1 mmol/L PMSF), and homogenized (50 strokes) in a dounce homogenizer. The homogenates were centrifuged for 10 minutes at 2000g; the resulting supernatant was centrifuged for 45 minutes at 30 000g. The plasma membranes were resuspended in HBSS containing a cocktail of protease inhibitors and analyzed by PAGE, electroblotting, and probing with an antibody to biotin (Sigma-Aldrich). Bands corresponding to biotin-labeled thiols were visualized with chemiluminescent detection.
Determination of Gene Expression Levels by Real-Time Polymerase Chain Reaction
Total cellular mRNA was isolated with TRIzol (Life Technologies) following the manufacturer’s protocol, and reverse transcription was performed to generate cDNAs (BD Biosciences). For quantitative real-time polymerase chain reaction (PCR), amplification was performed in triplicate on an iCycler IQ Multicolor Real-Time PCR Detection System (Biorad) for 35 cycles as follows: 95°C for 30 seconds, 62°C for 30 seconds, and 72°C for 1 minute. Quantification and melting curves were analyzed with iCycler software normalized to the ;-actin.
Results
Thiol/Disulfide Redox States in Human Plasma
No Effect of Oxidized Extracellular Cys/CySS Redox State on Cellular GSH/GSSG Redox
Initial extracellular Eh values for Cys/CySS in the culture medium calculated from measured Cys and CySS concentrations were comparable to the intended values (the Table). The extracellular redox states of Cys/CySS changed as a function of time, with the more reduced value becoming oxidized and the more oxidized value becoming more reduced over a 10-hour time course (Figure 2). In contrast, there was no change in cellular GSH/GSSG redox state (the Table), a finding consistent with recent studies showing that Cys/CySS and GSH/GSSG redox are maintained independently.10 Although the extracellular redox states changed with time, the results show that the model is adequate to test whether an oxidized extracellular Cys/CySS redox condition affects cells differently than a more reduced redox state.
No Changes in Cellular GSH/GSSG Redox State by Variation in Extracellular Cys/CySS Redox State
Oxidized Extracellular Cys/CySS Redox State Stimulates Monocytes Adhesion to Vascular Endothelium
The effect of oxidative extracellular Cys/CySS redox state on cell-cell adhesion was examined with an inverted microscope (Figure 3A) and fluorescence examination (Figure 3B and 3C) from independent experiments. BAECs exposed to Cys/CySS redox state of Eh, 0 mV for 36 hours with medium changed every 10 hours, resulted in a significant increase in attachment of monocyte THP1 cells to BAECs (Figure 3A). In the 0-mV panel of Figure 3A, dark and round images are THP1 cells; BAECs are the lighter, hazy background cells seen in the –150-mV panel. Quantification of adherent THP1 cells on BAECs in 96-well plates showed that incubation of BAECs at 0 mV stimulated attachment of monocytes to BAECs (2.8-fold increase at 0 mV to that of –150 mV in Figure 3B). Additionally, we examined whether extracellular Cys/CySS redox state has an effect on THP1 monocytes. THP1 cells were exposed to the extracellular Cys/CySS redox potential for 36 hours, followed by labeling with calcein-AM, and added to BAECs for cell-cell adhesion. Similar to the results obtained by BAECs exposed to the extracellular Cys/CySS redox potential, THP1 also stimulated attachment to BAECs 2.2-fold at Eh, 0 mV compared with that at –150 mV (Figure 3C). Thus, exposure of either BAECs or THP1 cells to more oxidized Cys/CySS redox state was sufficient to stimulate increased THP1 adhesion to BAEC.
Transcriptional Levels of Adhesion Molecules ICAM-1, PECAM-1, and P-selectin in BAECs Were Enhanced by Oxidative Extracellular Cys/CySS Redox Potential
Increased cell adhesion in atherosclerosis is mediated by increased expression of adhesion molecules in endothelial cells. To determine the influence of extracellular thiol/disulfide redox potential on expression of adhesion molecules, transcriptional regulation of the members of the cell adhesion molecules and the selectin family, including ICAM-1, PECAM-1, P-selectin, and E-selectin, was examined with real-time PCR. Messenger RNA levels for ICAM-1, PECAM-1, and P-selectin were increased in cells exposed to the more oxidized extracellular Eh (Figure 4). The maximal increase in gene expression was 2-fold for ICAM-1 (Figure 4A), 2-fold for PECAM-1 (Figure 4B), and 2.3-fold for P-selectin (Figure 4C) at Eh, 0 mV compared with that at –150 mV. There was a 2.3-fold increase in E-selectin at –80 mV (Figure 4D) but not at 0 mV. Collectively, the data show that exposure of endothelial cells to oxidized extracellular Cys/CySS redox state increases mRNA expression for cell-cell adhesion molecules.
NF-B Transcriptional Activation by Oxidative Extracellular Cys/CySS Redox State
NF-B is a redox-sensitive transcription factor that functions in regulation of adhesion molecule expression. To monitor NF-B activation by extracellular Cys/CySS redox potential, BAECs were cotransfected with the NF-B reporter plasmids. NF-B luciferase activity was increased 1.7-fold at Eh, 0 mV compared with Eh, –150 mV (Figure 5, bar graph) with no significant change at Eh, –80 mV. We further examined degradation of IB by Western blot analysis, probing with antibodies raised against total IB. Degradation of IB occurred at 0 mV, consistent with the results of enhanced NF-B luciferase activity at Eh, 0 mV (Figure 5, top). These results show that the oxidation of extracellular Cys/CySS redox state activates NF-B, thereby suggesting that increased expression of adhesion molecules in response to oxidized extracellular Eh could be mediated by NF-B.
Oxidative Extracellular Cys/CySS Redox Potential Stimulates Intracellular H2O2 but Not NO Formation by Mediation of Plasma Membrane Thiols
NF-B activation is mediated by oxidative events in the cytoplasm, yet data provided above show that oxidized extracellular Eh does not result in detectable oxidation of cellular GSH/GSSG redox state. Consequently, we examined whether oxidative extracellular redox potential was associated with increased H2O2 generation as measured by oxidative conversion of nonfluorescent DCFH to fluorescent DCF and by chemiluminescence enhanced by luminol oxidation. DCF fluorescence was elevated 1.5-fold and 1.2-fold in BAECs exposed to Eh, 0 mV and Eh, –80 mV for 2 hours, respectively, compared with that of Eh, –150 mV (Figure 6B through 6D). After 36 hours, the increase was 2.5- and 1.5-fold at 0 and –80 mV, respectively (data not shown). In addition to the measurement of intracellular ROS by DCF fluorescence, both intracellular ROS and extracellular ROS were examined by luminol-enhanced chemiluminescence using BAECs exposed with redox treatment for 2 hour. Chemiluminescence by extracellular Eh demonstrated a significant increase in cells exposed to Eh, 0 mV and –80 mV compared with that of Eh, –150 mV (1.3±0.024 and 1.2±0.03 at 0 and –80 mV, respectively; Figure 6A). Fold increase of chemiluminescence was also similarly elevated when cells were exposed to redox for 36 hours (data not shown).
To determine whether the increase in DCF fluorescence by oxidized extracellular Eh was due to H2O2 production, BAECs were preincubated with membrane-permeable catalase-PEG at a concentration of 0, 50, 100, and 500 U/mL before being exposed to extracellular Eh for 2 hours, followed by DCF analysis (Figure 6B). Catalase expression in BAECs inhibited DCF fluorescence dose dependently, suggesting that the DCF fluorescence increase was due to H2O2 production. To indicate involvement of a flavo protein such as an NADPH oxidase as an enzyme responsible for H2O2 production, BAECs were preincubated with the flavin-dependent oxygenase inhibitor DPI at 5 μmol/L for 1 hour before redox treatment (Figure 6C). DPI inhibited H2O2 production, suggesting that NADPH oxidase mediates oxidative Cys/CySS Eh-dependent H2O2 production. Additionally, we examined the possible role of NO in the extracellular Cys/CySS redox–elicited response by pretreating BAECs with the NOS inhibitors L-NAME and L-NNA and analyzing DCF fluorescence increase after exposing cells to extracellular Eh for 2 hours (Figure 6D). Inhibition of endothelial NOS had no effect on DCF increase in response to extracellular Eh, indicating that NO is not involved in the oxidative extracellular Eh-dependent H2O2 production. To determine whether intracellular responses to extracellular Eh were regulated by plasma membrane thiols, BAECs were pretreated with the non–cell-permeable thiol-reacting reagents AMS and qBBr before exposing cells with appropriate redox potential. qBBr and AMS are bimane and maleimide derivatives, respectively, that are readily conjugated to thiols to block thiol/disulfide interactions. qBBr is positively charged; AMS is negatively charged. The high polarity decreases membrane permeability and makes these reagents useful for determining whether redox-sensitive sites are exposed at the extracellular sites. Pretreating BAECs with AMS or qBBr (0.5 mmol/L) for 1 hour inhibited oxidative extracellular redox potential–induced increases in H2O2 production (Figure 7A). Additionally, cell surface thiols were quantified after exposure to extracellular Eh by monitoring fluorescence of AMS-bound thiols. Thiols were detected more at reduced redox potential than at oxidized state (1.3- and 1.2-fold at –150 and –80 mV, respectively), suggesting that extracellular Cys/CySS redox potential plays a role in regulating thiol/disulfide redox states of extracellular membrane proteins. In addition, protein thiols in the extracellular membrane were examined by Western blot analysis using thiol-reactive biotinylated reagent (Figure 7C), demonstrating more thiols detected at –150 mV than –80 or 0 mV, which was consistent with Figure 7B. Thus, these results show that key redox events of the inflammatory processes associated with atherosclerosis are controlled by the redox state of Cys/CySS at the extracellular surface of endothelial cells.
Discussion
Accumulating evidence shows that plasma GSH/GSSG redox and/or Cys/CySS redox state is oxidized in association with aging,18,23 cigarette smoking,19 type 2 diabetes,24 and high-dose chemotherapy.25 The GSH/GSSG and Cys/CySS pools are not in equilibrium in plasma; however, in this in vitro model, distinct extracellular GSH/GSSG and Cys/CySS redox states are not maintained. Thus, it is not possible to attribute effects explicitly to the GSH/GSSG or Cys/CySS pools. This means that the prevention of ROS production and cell adhesion at –150 mV could be mediated by the low concentration of GSH exported from cells, which would be maintained at a more reduced state by Cys/CySS redox at –150 than 0 mV, or it could be an effect due directly to the redox state of the added Cys/CySS. More importantly, the conditions of our study suggest that humans without known risk factors for cardiovascular disease may have plasma Cys/CySS redox levels that are in the range to significantly activate the mechanisms studied. In unpublished studies of acrolein, a biological aldehyde produced during lipid peroxidation and linked to cardiovascular disease development, atherosclerotic events, including ROS production and monocyte adhesion to endothelium, were markedly enhanced with oxidative extracellular Cys/CySS redox potential (data not shown). This result supports the idea that extracellular Cys/CySS redox stress could amplify the relatively small (but significant) changes observed with aging and smoking into the range that promotes the development of atherosclerosis.
The finding that extracellular thiol/disulfide redox controls signaling in endothelial cells is consistent with previous findings that cell surface thiols have critical signaling functions, eg, in EGFR, PDGFR, IGFR, NMDA receptor, and other ion channels and transporters.26–28 In studies to detect proteins that undergo oxidation as thiol/disulfide redox state is changed from –150 to 0 mV, we found that several cell surface proteins are oxidized (Figure 7C). Together with the finding that reaction of nonpermeant alkylating agents to the plasma membrane thiols causes inhibition of Cys/CySS-redox dependent H2O2 production, our results suggest that specific protein sensors are present on the cell surface that signal responses to extracellular thiol/disulfide redox state. Accordingly, to initiate inflammatory signaling events, surface receptor(s) responsible for the changes in extracellular Cys/CySS redox may not need a prolonged oxidative redox environment. In fact, H2O2 production was detectable for 2 hours of exposure of oxidative extracellular Eh (Figure 6), although the fold increase in H2O2 was relatively less than that of 36 hours, as mentioned earlier. However, to determine late responses elicited by oxidative Eh such as elevation of cell adhesion molecules and subsequent increases in monocytes adhesion to BAECs, cells were required for extended incubation over 24 hours. The data imply that events that could induce oxidation of thiol/disulfide redox such as low sulfur amino acid intake, alcohol consumption, or viral infection could have significant effects on cell adhesion.
Previous studies of the consequences of variation in extracellular thiol/disulfide redox state show that proliferation of the colon cancer cell line Caco2 is redox dependent, with the rate of BrdU incorporation at –150 mV being 2-fold greater than at 0 mV. This redox-dependent growth effect occurred without a detectable difference in intracellular GSH/GSSG redox.11 Addition of IGF-1, EGF, KGF, or glutamine resulted in stimulation of cell growth under more oxidizing conditions but not under the most reduced extracellular redox state,11 indicating that extracellular redox-dependent effects may be mediated by growth factor receptors. Consistent with previous studies, the present study shows that the oxidative extracellular Cys/CySS Eh-elicited inflammatory event is not associated with oxidation of intracellular GSH/GSSG redox potential, although H2O2 is generated under this condition. Previously, we found that H2O2 signaling to activate mitogen-activated protein kinase pathways can occur without oxidation of cellular GSH or thioredoxin-1.20 This finding supports the theory that the extracellular Cys/CySS Eh can function as a signaling regulator to induce intracellular inflammatory signaling in the absence of global oxidation of the intracellular thiol/disulfide GSH/GSSG.
Metal-catalyzed autoxidation of sulfhydryl compounds, including GSH and Cys, is accompanied by production of superoxide,29 and the rate of this reaction increases with pH >7.8.30 However, our data showed that the oxidative Cys/CySS redox potential (0 mV: CySS, 99.7 μmol/L; Cys, 0.6 μmol/L) augmented H2O2 production more than reduced redox potential (–150 mV: Cys, 175 μmol/L; CySS, 12.5 μmol/L). In addition, variations in Cys and CySS concentrations maintaining the same redox potential with 100 μmol/L total Cys equivalents instead of 200 μmol/L did not affect the enhancement of monocyte adhesions to BAECs or H2O2 production (data not shown). Thus, the enhanced H2O2 generation at 0 mV cannot be explained by autoxidation of thiols in the culture medium.
The in vitro cell culture model used in the present study relied on controlled variations in cysteine/cystine redox state in culture medium. Because of the artificial nature of cell culture medium compared with plasma, it remains unclear whether the response of cells in vivo would be qualitatively or quantitatively the same as reported here. Additional in vitro studies with controlled redox state in plasma or in vivo studies are needed to address this important issue.
In summary, our results support a novel mechanism of inflammatory signaling in early atherosclerosis. Oxidation of extracellular thiol/disulfide redox state oxidizes plasma membrane protein thiols, stimulates H2O2 production in the signaling pathway, and results in NF-B activation, enhanced expression of cell-cell adhesion molecules, and attachment of monocytes to endothelial cells. Inhibition of H2O2 generation by pretreatment with non–cell-permeable alkylating reagents suggests that plasma membrane proteins that are sensitive to extracellular Cys/CySS redox state may provide novel targets for protection against the pathogenesis of atherosclerosis. Moreover, the results suggest that plasma thiol/disulfide redox state may provide a useful, early marker of atherosclerosis development and that nutritional and therapeutic means to normalize oxidized redox state could provide a strategy to protect against disease development.
Acknowledgments
This work was supported by NIH grant ES011195. We appreciate the critical comments provided by Dr David G. Harrison (Emory University).
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Abstract
Background— Oxidative stress, a contributing factor to atherosclerosis, causes oxidation of biological thiols, which can be quantified in terms of the thiol/disulfide redox. The major thiol/disulfide redox couple in human plasma is cysteine (Cys) and its disulfide, cystine (CySS). Although atherosclerosis has previously been associated with Cys/CySS oxidation, whether oxidation of Cys/CySS contributes in a causal way to atherosclerosis development is not known. We examined the function of extracellular Cys/CySS redox potential (Eh) in the regulation of early events of atherosclerosis using cultured aortic endothelial cells and monocytes as a vascular model system.
Methods and Results— To determine the range of thiol/disulfide redox state in human plasma, we analyzed levels of Cys, CySS, glutathione (GSH), and glutathione disulfide (GSSG) and calculated Eh according to the Nernst equation. Eh of Cys/CySS and GSH/GSSG was –120 to –20 and –200 to –50 mV, respectively. To approximate this range, endothelial cells were exposed to initial Eh from –150 mV (most reduced) to 0 mV (most oxidized). Compared with more reduced Eh, oxidized Eh of Cys/CySS stimulated H2O2 but not nitric oxide production, activated nuclear factor-B, increased expression of adhesion molecules (intercellular adhesion molecule-1, platelet endothelial cell adhesion molecule-1, P-selectin), and stimulated monocytes binding to endothelial cells. Extracellular Eh regulated thiol/disulfide redox states of extracellular membrane proteins and H2O2 production, indicating that variation in extracellular Eh is detected and signaled at the cell surface.
Conclusions— The extracellular thiol/disulfide Eh of the Cys/CySS couple plays a key role in regulating early events of atherosclerosis and could be useful as a potential marker for vascular disease risk.
Key Words: atherosclerosis ; cell adhesion molecules ; endothelium ; inflammation ; plasma
Introduction
Considerable evidence implicates oxidative stress and endothelial dysfunction and injury as initiation events leading to atherosclerotic cardiovascular disease.1–4 Atherosclerosis is a chronic immune-mediated disease; its initiation, progression, and destabilization are driven and regulated by inflammatory cells. Critical events in the initiation of the vascular inflammatory disease are the adhesion of leukocytes to the dysfunctional and injured endothelium and the penetration of these cells into the vessel wall to generate foam cells. Mechanistic studies show that transcriptional activation of adhesion molecules, including P-selectin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule (PECAM),5–7 by nuclear factor-B (NF-B)8,9 plays a pivotal role in the process of cell-cell adhesion. NF-B is activated by oxidative stress, which can be caused by reactive oxygen species (ROS) and reactive nitrogen species generated in free radical reactions from products of enzymes (NADPH oxidases and nitric oxide [NO] synthases [NOSs]) present in the vascular endothelium and smooth muscle.
Accumulating evidence indicates that redox-regulated biological signaling pathways respond not only to ROS but also to the redox state (Eh) of the thiol/disulfide couple.10–12 The reversible redox reactions of thiol/disulfide pools regulate diverse biological processes, including enzyme catalysis, gene expression, cell proliferation, and atherosclerosis.13–16 Most studies thus far have focused on the major cellular thiol, glutathione (GSH), and its disulfide (GSSG). Relatively less information is available concerning cysteine (Cys) and its disulfide, cystine (CySS), which together constitute the predominant low-molecular-weight thiol/disulfide pool in human plasma. Cys residues in proteins are critical for the function of many enzymes, receptors, ion channels, transporters, and transcription factors and are common targets of oxidation. Free cysteine undergoes rapid autoxidation and is toxic to mammalian systems at concentrations exceeding the normal, low-micromolar concentrations found in plasma because its oxidation results in generation of ROS. However, little is known about the effects of variation in Cys/CySS redox state, such as the progressive oxidation of Cys/CySS associated with increasing age17,18 and smoking,19 on cellular events related to atherosclerosis.
In the present study, we hypothesized that oxidation of the thiol/disulfide redox state over the range found in vivo in human plasma is a key determinant of early events of vascular disease development. To test this hypothesis, we compiled data from 4 previous studies of thiol/disulfide redox state in human plasma to determine the range of redox potential. Initial redox states spanning this range were then used with an in vitro model system to study early signaling events of monocyte adhesion to vascular endothelial cells. Results show that oxidized extracellular redox state of Cys/CySS (Eh, 0 mV) stimulated attachment of monocytes through mechanisms involving stimulation of H2O2 generation, activation of NF-B, and increased expression of adhesion molecules, including ICAM-1, PECAM-1, and P-selectin. These findings suggest that oxidation of extracellular Cys/CySS redox potential could play a key role in proatherosclerosis processes.
Methods
Cell Culture, Transfection, and Cell Treatment
Bovine aortic endothelial cells (BAECs) harvested from descending thoracic aortas and THP1 purchased from ATCC were maintained with 10% FBS (37°C, 5% CO2) in DMEM and RPMI, respectively. Before experiments with controlled initial redox states, medium was changed to cyst(e)ine-free DMEM or cyst(e)ine-free RPMI with 0.5% FBS. For transfections, BAECs (4 to 5x105 cells per plate) were grown overnight and transfected with FuGene 6 (Roche). Cells were exposed to different Eh for 1 day before the luciferase assay was performed. For transfections, cells were cotransfected with pNFB-Luc (BD science) or pTAL-Luc (BD science) and with pcDNA3.1 containing lacZ gene (Invitrogen). We obtained 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, disodium salt (AMS), monobromotrimethylammoniobimane bromide (qBBr), biotin-maleimide [N-(3-maleimidylpropionyl)biocytin], 6-carboxy-2', 7'-dichlorofluorescin diacetate (DCF-DA), and luminol from Molecular Probes; diphenyleneiodonium chlroride (DPI) was from Biolmol. Cys, CySS, catalase–polyethylene glycol (PEG), NG-nitro-L-arginine methyl ester hydrochloride (L-NAME), and NG-nitro-L-arginine (L-NNA) were from Sigma-Aldrich.
Assays
The luciferase activity was normalized for transfection efficiency as described previously.20 To generate the desired Eh, varied concentrations of Cys and CySS were added to cyst(e)ine-free media to give a constant total amount of Cys equivalents (200 μmol/L) as described previously.11 Cells were replenished with fresh medium every 10 hours. The medium and cell lysates collected for the analyses of Cys, CySS, GSH, and GSSG were derivatized, followed by high-performance liquid chromatography analysis as reported previously.15 Eh for Cys/CySS was calculated with the Nernst equation: Eh=E0+RT/2F ln([CySS]/[Cys]2), –250 mV for E0 at pH 7.4.15 To examine the effect of Cys/CySS redox on adherence of monocytes to BAECs, THP1 cells labeled by calcein AM (Molecular Probes)21 were added to BAECs at a cell density 1x106 or 5x104 per well for 6- or 96-well plates, respectively. The 96-well plates were incubated for 30 minutes (37°C, 5% CO2), washed 3 times to remove unbound THP1, and scanned by the plate reader to measure total fluorescence in each well. For ROS detection, dichlorofluorescin (DCFH) and luminol were used. To measure ROS production by DCFH oxidation, BAECs plated into 96-well plate were incubated with the Cys/CySS-limiting medium. BAECs were washed with KRH buffer,22 incubated with DCFH-DA at 50 μmol/L for 30 minutes (37°C, 5% CO2), and washed, and the fluorescence of cells from each well was measured by the plate reader. Additionally, ROS production in BAECs after Cys/CySS redox treatment was determined by monitoring chemiluminescence enhanced by luminol.
Determination of Extracellular Thiols and Western Blot Analysis
To determine extracellular thiols, BAECs plated in 96-well plate were exposed to Cys/CySS redox for 2 hours, washed twice with Hank’s balanced salt solution (HBSS), incubated with AMS (0.5 mmol/L) for 1 hour, and washed, and extracellular thiols conjugated with AMS were quantified by fluorescence (excitation, 324 nm; emission, 410 nm). To examine extracellular thiols present in the plasma membrane by Western blot analysis, BAECs grown in 150-mm plates were exposed to Cys/CySS redox for 2 hours, washed with HBSS, incubated with thiol-reactive biotinylation reagent (biotin maleimide, 0.5 mmol/L) for 1 hour, washed, lysed with a lysis buffer (1 mmol/L NaHCO3, pH 7.2, 2 mmol/L CaCl2, 5 mmol/L MgCl2, 1 mmol/L PMSF), and homogenized (50 strokes) in a dounce homogenizer. The homogenates were centrifuged for 10 minutes at 2000g; the resulting supernatant was centrifuged for 45 minutes at 30 000g. The plasma membranes were resuspended in HBSS containing a cocktail of protease inhibitors and analyzed by PAGE, electroblotting, and probing with an antibody to biotin (Sigma-Aldrich). Bands corresponding to biotin-labeled thiols were visualized with chemiluminescent detection.
Determination of Gene Expression Levels by Real-Time Polymerase Chain Reaction
Total cellular mRNA was isolated with TRIzol (Life Technologies) following the manufacturer’s protocol, and reverse transcription was performed to generate cDNAs (BD Biosciences). For quantitative real-time polymerase chain reaction (PCR), amplification was performed in triplicate on an iCycler IQ Multicolor Real-Time PCR Detection System (Biorad) for 35 cycles as follows: 95°C for 30 seconds, 62°C for 30 seconds, and 72°C for 1 minute. Quantification and melting curves were analyzed with iCycler software normalized to the ;-actin.
Results
Thiol/Disulfide Redox States in Human Plasma
No Effect of Oxidized Extracellular Cys/CySS Redox State on Cellular GSH/GSSG Redox
Initial extracellular Eh values for Cys/CySS in the culture medium calculated from measured Cys and CySS concentrations were comparable to the intended values (the Table). The extracellular redox states of Cys/CySS changed as a function of time, with the more reduced value becoming oxidized and the more oxidized value becoming more reduced over a 10-hour time course (Figure 2). In contrast, there was no change in cellular GSH/GSSG redox state (the Table), a finding consistent with recent studies showing that Cys/CySS and GSH/GSSG redox are maintained independently.10 Although the extracellular redox states changed with time, the results show that the model is adequate to test whether an oxidized extracellular Cys/CySS redox condition affects cells differently than a more reduced redox state.
No Changes in Cellular GSH/GSSG Redox State by Variation in Extracellular Cys/CySS Redox State
Oxidized Extracellular Cys/CySS Redox State Stimulates Monocytes Adhesion to Vascular Endothelium
The effect of oxidative extracellular Cys/CySS redox state on cell-cell adhesion was examined with an inverted microscope (Figure 3A) and fluorescence examination (Figure 3B and 3C) from independent experiments. BAECs exposed to Cys/CySS redox state of Eh, 0 mV for 36 hours with medium changed every 10 hours, resulted in a significant increase in attachment of monocyte THP1 cells to BAECs (Figure 3A). In the 0-mV panel of Figure 3A, dark and round images are THP1 cells; BAECs are the lighter, hazy background cells seen in the –150-mV panel. Quantification of adherent THP1 cells on BAECs in 96-well plates showed that incubation of BAECs at 0 mV stimulated attachment of monocytes to BAECs (2.8-fold increase at 0 mV to that of –150 mV in Figure 3B). Additionally, we examined whether extracellular Cys/CySS redox state has an effect on THP1 monocytes. THP1 cells were exposed to the extracellular Cys/CySS redox potential for 36 hours, followed by labeling with calcein-AM, and added to BAECs for cell-cell adhesion. Similar to the results obtained by BAECs exposed to the extracellular Cys/CySS redox potential, THP1 also stimulated attachment to BAECs 2.2-fold at Eh, 0 mV compared with that at –150 mV (Figure 3C). Thus, exposure of either BAECs or THP1 cells to more oxidized Cys/CySS redox state was sufficient to stimulate increased THP1 adhesion to BAEC.
Transcriptional Levels of Adhesion Molecules ICAM-1, PECAM-1, and P-selectin in BAECs Were Enhanced by Oxidative Extracellular Cys/CySS Redox Potential
Increased cell adhesion in atherosclerosis is mediated by increased expression of adhesion molecules in endothelial cells. To determine the influence of extracellular thiol/disulfide redox potential on expression of adhesion molecules, transcriptional regulation of the members of the cell adhesion molecules and the selectin family, including ICAM-1, PECAM-1, P-selectin, and E-selectin, was examined with real-time PCR. Messenger RNA levels for ICAM-1, PECAM-1, and P-selectin were increased in cells exposed to the more oxidized extracellular Eh (Figure 4). The maximal increase in gene expression was 2-fold for ICAM-1 (Figure 4A), 2-fold for PECAM-1 (Figure 4B), and 2.3-fold for P-selectin (Figure 4C) at Eh, 0 mV compared with that at –150 mV. There was a 2.3-fold increase in E-selectin at –80 mV (Figure 4D) but not at 0 mV. Collectively, the data show that exposure of endothelial cells to oxidized extracellular Cys/CySS redox state increases mRNA expression for cell-cell adhesion molecules.
NF-B Transcriptional Activation by Oxidative Extracellular Cys/CySS Redox State
NF-B is a redox-sensitive transcription factor that functions in regulation of adhesion molecule expression. To monitor NF-B activation by extracellular Cys/CySS redox potential, BAECs were cotransfected with the NF-B reporter plasmids. NF-B luciferase activity was increased 1.7-fold at Eh, 0 mV compared with Eh, –150 mV (Figure 5, bar graph) with no significant change at Eh, –80 mV. We further examined degradation of IB by Western blot analysis, probing with antibodies raised against total IB. Degradation of IB occurred at 0 mV, consistent with the results of enhanced NF-B luciferase activity at Eh, 0 mV (Figure 5, top). These results show that the oxidation of extracellular Cys/CySS redox state activates NF-B, thereby suggesting that increased expression of adhesion molecules in response to oxidized extracellular Eh could be mediated by NF-B.
Oxidative Extracellular Cys/CySS Redox Potential Stimulates Intracellular H2O2 but Not NO Formation by Mediation of Plasma Membrane Thiols
NF-B activation is mediated by oxidative events in the cytoplasm, yet data provided above show that oxidized extracellular Eh does not result in detectable oxidation of cellular GSH/GSSG redox state. Consequently, we examined whether oxidative extracellular redox potential was associated with increased H2O2 generation as measured by oxidative conversion of nonfluorescent DCFH to fluorescent DCF and by chemiluminescence enhanced by luminol oxidation. DCF fluorescence was elevated 1.5-fold and 1.2-fold in BAECs exposed to Eh, 0 mV and Eh, –80 mV for 2 hours, respectively, compared with that of Eh, –150 mV (Figure 6B through 6D). After 36 hours, the increase was 2.5- and 1.5-fold at 0 and –80 mV, respectively (data not shown). In addition to the measurement of intracellular ROS by DCF fluorescence, both intracellular ROS and extracellular ROS were examined by luminol-enhanced chemiluminescence using BAECs exposed with redox treatment for 2 hour. Chemiluminescence by extracellular Eh demonstrated a significant increase in cells exposed to Eh, 0 mV and –80 mV compared with that of Eh, –150 mV (1.3±0.024 and 1.2±0.03 at 0 and –80 mV, respectively; Figure 6A). Fold increase of chemiluminescence was also similarly elevated when cells were exposed to redox for 36 hours (data not shown).
To determine whether the increase in DCF fluorescence by oxidized extracellular Eh was due to H2O2 production, BAECs were preincubated with membrane-permeable catalase-PEG at a concentration of 0, 50, 100, and 500 U/mL before being exposed to extracellular Eh for 2 hours, followed by DCF analysis (Figure 6B). Catalase expression in BAECs inhibited DCF fluorescence dose dependently, suggesting that the DCF fluorescence increase was due to H2O2 production. To indicate involvement of a flavo protein such as an NADPH oxidase as an enzyme responsible for H2O2 production, BAECs were preincubated with the flavin-dependent oxygenase inhibitor DPI at 5 μmol/L for 1 hour before redox treatment (Figure 6C). DPI inhibited H2O2 production, suggesting that NADPH oxidase mediates oxidative Cys/CySS Eh-dependent H2O2 production. Additionally, we examined the possible role of NO in the extracellular Cys/CySS redox–elicited response by pretreating BAECs with the NOS inhibitors L-NAME and L-NNA and analyzing DCF fluorescence increase after exposing cells to extracellular Eh for 2 hours (Figure 6D). Inhibition of endothelial NOS had no effect on DCF increase in response to extracellular Eh, indicating that NO is not involved in the oxidative extracellular Eh-dependent H2O2 production. To determine whether intracellular responses to extracellular Eh were regulated by plasma membrane thiols, BAECs were pretreated with the non–cell-permeable thiol-reacting reagents AMS and qBBr before exposing cells with appropriate redox potential. qBBr and AMS are bimane and maleimide derivatives, respectively, that are readily conjugated to thiols to block thiol/disulfide interactions. qBBr is positively charged; AMS is negatively charged. The high polarity decreases membrane permeability and makes these reagents useful for determining whether redox-sensitive sites are exposed at the extracellular sites. Pretreating BAECs with AMS or qBBr (0.5 mmol/L) for 1 hour inhibited oxidative extracellular redox potential–induced increases in H2O2 production (Figure 7A). Additionally, cell surface thiols were quantified after exposure to extracellular Eh by monitoring fluorescence of AMS-bound thiols. Thiols were detected more at reduced redox potential than at oxidized state (1.3- and 1.2-fold at –150 and –80 mV, respectively), suggesting that extracellular Cys/CySS redox potential plays a role in regulating thiol/disulfide redox states of extracellular membrane proteins. In addition, protein thiols in the extracellular membrane were examined by Western blot analysis using thiol-reactive biotinylated reagent (Figure 7C), demonstrating more thiols detected at –150 mV than –80 or 0 mV, which was consistent with Figure 7B. Thus, these results show that key redox events of the inflammatory processes associated with atherosclerosis are controlled by the redox state of Cys/CySS at the extracellular surface of endothelial cells.
Discussion
Accumulating evidence shows that plasma GSH/GSSG redox and/or Cys/CySS redox state is oxidized in association with aging,18,23 cigarette smoking,19 type 2 diabetes,24 and high-dose chemotherapy.25 The GSH/GSSG and Cys/CySS pools are not in equilibrium in plasma; however, in this in vitro model, distinct extracellular GSH/GSSG and Cys/CySS redox states are not maintained. Thus, it is not possible to attribute effects explicitly to the GSH/GSSG or Cys/CySS pools. This means that the prevention of ROS production and cell adhesion at –150 mV could be mediated by the low concentration of GSH exported from cells, which would be maintained at a more reduced state by Cys/CySS redox at –150 than 0 mV, or it could be an effect due directly to the redox state of the added Cys/CySS. More importantly, the conditions of our study suggest that humans without known risk factors for cardiovascular disease may have plasma Cys/CySS redox levels that are in the range to significantly activate the mechanisms studied. In unpublished studies of acrolein, a biological aldehyde produced during lipid peroxidation and linked to cardiovascular disease development, atherosclerotic events, including ROS production and monocyte adhesion to endothelium, were markedly enhanced with oxidative extracellular Cys/CySS redox potential (data not shown). This result supports the idea that extracellular Cys/CySS redox stress could amplify the relatively small (but significant) changes observed with aging and smoking into the range that promotes the development of atherosclerosis.
The finding that extracellular thiol/disulfide redox controls signaling in endothelial cells is consistent with previous findings that cell surface thiols have critical signaling functions, eg, in EGFR, PDGFR, IGFR, NMDA receptor, and other ion channels and transporters.26–28 In studies to detect proteins that undergo oxidation as thiol/disulfide redox state is changed from –150 to 0 mV, we found that several cell surface proteins are oxidized (Figure 7C). Together with the finding that reaction of nonpermeant alkylating agents to the plasma membrane thiols causes inhibition of Cys/CySS-redox dependent H2O2 production, our results suggest that specific protein sensors are present on the cell surface that signal responses to extracellular thiol/disulfide redox state. Accordingly, to initiate inflammatory signaling events, surface receptor(s) responsible for the changes in extracellular Cys/CySS redox may not need a prolonged oxidative redox environment. In fact, H2O2 production was detectable for 2 hours of exposure of oxidative extracellular Eh (Figure 6), although the fold increase in H2O2 was relatively less than that of 36 hours, as mentioned earlier. However, to determine late responses elicited by oxidative Eh such as elevation of cell adhesion molecules and subsequent increases in monocytes adhesion to BAECs, cells were required for extended incubation over 24 hours. The data imply that events that could induce oxidation of thiol/disulfide redox such as low sulfur amino acid intake, alcohol consumption, or viral infection could have significant effects on cell adhesion.
Previous studies of the consequences of variation in extracellular thiol/disulfide redox state show that proliferation of the colon cancer cell line Caco2 is redox dependent, with the rate of BrdU incorporation at –150 mV being 2-fold greater than at 0 mV. This redox-dependent growth effect occurred without a detectable difference in intracellular GSH/GSSG redox.11 Addition of IGF-1, EGF, KGF, or glutamine resulted in stimulation of cell growth under more oxidizing conditions but not under the most reduced extracellular redox state,11 indicating that extracellular redox-dependent effects may be mediated by growth factor receptors. Consistent with previous studies, the present study shows that the oxidative extracellular Cys/CySS Eh-elicited inflammatory event is not associated with oxidation of intracellular GSH/GSSG redox potential, although H2O2 is generated under this condition. Previously, we found that H2O2 signaling to activate mitogen-activated protein kinase pathways can occur without oxidation of cellular GSH or thioredoxin-1.20 This finding supports the theory that the extracellular Cys/CySS Eh can function as a signaling regulator to induce intracellular inflammatory signaling in the absence of global oxidation of the intracellular thiol/disulfide GSH/GSSG.
Metal-catalyzed autoxidation of sulfhydryl compounds, including GSH and Cys, is accompanied by production of superoxide,29 and the rate of this reaction increases with pH >7.8.30 However, our data showed that the oxidative Cys/CySS redox potential (0 mV: CySS, 99.7 μmol/L; Cys, 0.6 μmol/L) augmented H2O2 production more than reduced redox potential (–150 mV: Cys, 175 μmol/L; CySS, 12.5 μmol/L). In addition, variations in Cys and CySS concentrations maintaining the same redox potential with 100 μmol/L total Cys equivalents instead of 200 μmol/L did not affect the enhancement of monocyte adhesions to BAECs or H2O2 production (data not shown). Thus, the enhanced H2O2 generation at 0 mV cannot be explained by autoxidation of thiols in the culture medium.
The in vitro cell culture model used in the present study relied on controlled variations in cysteine/cystine redox state in culture medium. Because of the artificial nature of cell culture medium compared with plasma, it remains unclear whether the response of cells in vivo would be qualitatively or quantitatively the same as reported here. Additional in vitro studies with controlled redox state in plasma or in vivo studies are needed to address this important issue.
In summary, our results support a novel mechanism of inflammatory signaling in early atherosclerosis. Oxidation of extracellular thiol/disulfide redox state oxidizes plasma membrane protein thiols, stimulates H2O2 production in the signaling pathway, and results in NF-B activation, enhanced expression of cell-cell adhesion molecules, and attachment of monocytes to endothelial cells. Inhibition of H2O2 generation by pretreatment with non–cell-permeable alkylating reagents suggests that plasma membrane proteins that are sensitive to extracellular Cys/CySS redox state may provide novel targets for protection against the pathogenesis of atherosclerosis. Moreover, the results suggest that plasma thiol/disulfide redox state may provide a useful, early marker of atherosclerosis development and that nutritional and therapeutic means to normalize oxidized redox state could provide a strategy to protect against disease development.
Acknowledgments
This work was supported by NIH grant ES011195. We appreciate the critical comments provided by Dr David G. Harrison (Emory University).
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